Rapid antimicrobial susceptibility test, based on an analysis of changes in morphology and growth pattern of a microbial cell under different concentrations of various antimicrobial agents, and automated cell image analysis system therefor

ABSTRACT

Provided are a rapid antimicrobial susceptibility test, based on an analysis of changes in morphology and growth pattern of a microbial cell under different concentrations of various antimicrobial agents, and an automated cell image analysis system therefor. The antimicrobial susceptibility test is rapidly performed based on an analysis of changes in morphology and growth pattern of a microbial cell under different concentrations of various antimicrobial agents, and this makes it possible to obtain highly reliable test results faster by six to seven times than the standard method recommended by Clinical and Laboratory Standards Institute (CLSI).

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Division of application Ser. No. 14/883,101, filed Oct. 14, 2015, which in turn claims priority under 35 U.S.C. § 119 to Korean Patent Application No. 10-2014-0138535, filed on Oct. 14, 2014, and U.S. Provisional Patent Application No. 62/067,409, filed on Oct. 22, 2014, the entire contents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Example embodiments of the inventive concept relate to a rapid antimicrobial susceptibility test, based on an analysis of changes in morphology and growth pattern of a microbial cell under different concentrations of various antimicrobial agents, and an automated cell image analysis system therefor. In particular, the antimicrobial susceptibility test is rapidly performed based on an analysis of changes in morphology and growth pattern of a microbial cell under different concentrations of various antimicrobial agents, and this makes it possible to obtain highly reliable test results faster by six to seven times than the standard method recommended by Clinical and Laboratory Standards Institute (CLSI).

An antimicrobial susceptibility test (AST) may be performed to estimate effects of antimicrobial agents that will be used for treatment of infectious diseases, and it may include examining whether there is susceptibility, based on a minimal inhibitory concentration (MIC).

A conventional method of testing a cell reaction to different drugs may include steps of disposing a cell in a liquid or solid medium, mixing a to-be-tested drug with the liquid medium (or disposing a paper disk, in which the drug is absorbed, on the solid medium) to react the drug with the cell, and measuring turbidity or absorbance representing an extent of a growth reaction of the cell to the drug. However, the conventional method is a statistical method of collecting information on many cells, not on a change of each cell, and thus, in order to obtain meaningful statistical results, the number of cells should be increased up to a specific value (typically, ten million per 1 ml or higher), and thus, it takes a long time to culture cells (typically, 16-24 hours). Furthermore, according to the conventional method, it is impossible to realize an observation of a change of each individual cell under a drug and an real-time observation of each motile cell real-time, and moreover, it takes a long time and great effort to test many drugs, because each drug should be injected by an individual injection process.

Furthermore, in the case where a solid medium is used for the AST method, a KB-test has a limitation on the number of samples that can be disposed on each medium plate, and thus, in order to perform an AST process for tens of different antimicrobial agents, many agar medium plates are required. Even when an automated system (e.g., VITEK system) capable of minimizing a test time is used for the AST process, a turbidity of bacteria should be increased up to a specific value, and thus, the AST process also suffers from a long test time (typically, 12 hours or more). In addition, since a test environment for the conventional method is different from a human body, the results may be largely different from the real phenomenon in the human body (Gregory G. Anderson, et al. (2003), “Intracellular Bacterial Biofilm-Like Pods in Urinary Tract Infections”, Science 301, 105; Gallo et al. (2011), “Demonstration of Bacillus cereus in Orthopaedic-Implant-Related Infection with Use of a Multi-Primer Polymerase Chain Reaction-Mass Spectrometric Assay”, J Bone Joint Surg Am, 93).

To overcome technical limitations of the conventional AST method, there have been proposed various methods, called rapid AST (RAST), which make it possible to observe the division of microbes at an initial step. For example, each of such known methods may include a step of measuring the number of microbial cells in micro-fluidic channel, measuring a rotation speed of a magnetic bead to estimate a weight of microbes, measuring a fluorescence signal associated with metabolic activity of microbial cells in liquid droplet, or calculating an area of an image, which is occupied by microbes, to estimate susceptibility to an antimicrobial agent.

Meanwhile, microbial reactions to antimicrobial agents are very heterogeneous and are specifically dependent on antimicrobial agent conditions, but the known methods are performed based on the observation for examining whether a microbe is growing. In other words, in the known methods, only the presence or absence of ‘growth’ of a microbial cell is observed, without consideration for various morphological changes (other than ‘growth’ or ‘no-growth’) of a microbial cell, which may occur under different conditions of antimicrobial agents, and thus, the known methods suffer from low reliability of AST results.

SUMMARY

To overcome these difficulties, the inventors have examined correlation between the changes in morphology and growth pattern of a microbial cell under different concentrations of various antimicrobial agents and the results of Broth Micro-dilution (BMD) experiment and have invented an accurate and rapid antimicrobial susceptibility test (AST) method based on the examination of the correlation. Here, the changes in morphology and growth pattern of a microbial cell under different concentrations of various antimicrobial agents are analyzed in the invented AST method, and in this sense, the invented AST method is different from the conventional method, in which only the presence or absence of the growth of a microbe is measured to determine antimicrobial susceptibility of the microbe to antimicrobial agents.

According to some aspects of the inventive concept, example embodiments of the inventive concept relate to an antimicrobial susceptibility (AST) method, based on an analysis of changes in morphology and growth pattern of a microbial cell under antimicrobial agents, and the AST method may include the steps of:

-   (a) reacting a microbe with an antimicrobial agent; -   (b) imaging a change in morphology and growth pattern of a microbial     cell over time; -   (c) classifying the change in morphology and growth pattern of the     microbial cell, based on the obtained image; and -   (d) determining whether the microbe is resistant or susceptible to     the antimicrobial agent, based on the classification on the change     in morphology and growth pattern of the microbial cell.

In the AST method according to the inventive concept, the microbial cell may include a single microbial cell or a group of microbial cells.

In the AST method according to the inventive concept, in step (c), the change in morphology and growth pattern of the microbial cell may be classified into dividing, no-change, filament formation, and swelling formation.

In the AST method according to the inventive concept, in step (d), when the change in morphology and growth pattern of the microbial cell is classified as the dividing, the microbe may be determined to be resistant to the antimicrobial agent, and when the change in morphology and growth pattern of the microbial cell is classified as one of the no-change, the filament formation, and the swelling formation, the microbe may be determined to be susceptible to the antimicrobial agent.

In the AST method according to the inventive concept, the microbe may include Enterococcus faecium, Staphylococcus aureus, Klebsiella species, Acinetobacter baumannii, Pseudomonas aeruginosa, or Enterobacter species.

In the AST method according to the inventive concept, the filament formation may include a microbial reaction, except for a reaction of gram-negative bacteria including Pseudomonas aeruginosa and Escherichia coli to penems of β-lactam antimicrobial agents.

In the AST method according to the inventive concept, the swelling formation may include a microbial reaction of gram-negative bacteria including Pseudomonas aeruginosa and Escherichia coli to penems of β-lactam antimicrobial agents.

According to other aspects of the inventive concept, example embodiments of the inventive concept relate to an antimicrobial susceptibility (AST) method, which may be performed based on an analysis of changes in morphology and growth pattern of a microbial cell under antimicrobial agents. For example, the AST method may include the steps of:

-   (a) reacting a microbe with an antimicrobial agent; -   (b) imaging a change in morphology and growth pattern of a microbial     cell over time; -   (c) inspecting an image obtained in step (b) to observe the change     in morphology and growth pattern of the microbial cell; and -   (d) determining an antimicrobial susceptibility of the microbe in     such a way that, when the microbial cell is observed to be in a     state of dividing, the microbial cell is determined to be resistant     to the antimicrobial agent, and when the microbial cell is observed     to be in a state of no-change, filament formation, or swelling     formation, the microbial cell is determined to be susceptible to the     antimicrobial agent.

According to still other aspects of the inventive concept, example embodiments of the inventive concept relate to an automated cell image analysis system configured to perform an antimicrobial susceptibility (AST) method, based on an analysis of changes in morphology and growth pattern of a microbial cell under antimicrobial agents, and the cell image analysis system may include:

-   (a) a culture chip for culture of a microbe and an antimicrobial     agent and for an imaging of a change in morphology and growth     pattern of a microbial cell; -   (b) an optical image analysis device filming a culturing region of     the microbial cell and detecting an image of the microbial cell; and -   (c) a reader analyzing the detected image of the microbial cell to     obtain information on a total area occupied by microbial cells, the     number of microbial cells, and a total length of microbial cells and     determining antimicrobial susceptibility of the microbial cell.

In the automated cell image analysis system for the AST method according to the inventive concept, the change in morphology and growth pattern of the microbe is classified into dividing, no-change, filament formation, and swelling formation, according to the total area occupied by microbial cells, the number of microbial cells, and the total length of microbial cells, and the antimicrobial susceptibility is determined based on the classification of the change in morphology and growth pattern of the microbe.

In the automated cell image analysis system for the AST method according to the inventive concept, the optical image analysis device may include a tungsten lamp, an LED device, or a laser light source, which serve as a visible light source for obtaining an optical image. The optical image analysis device may include a charge coupled device (CCD) or complementary metal oxide semiconductor (CMOS) camera for obtaining a cell image transmitted through an objective lens.

The use of the AST method according to the inventive concept (i.e., based on an analysis of changes in morphology and growth pattern of a microbial cell under antimicrobial agents) makes it possible to obtain a more accurate AST result, compared with that of the conventional AST method, as will be described with reference to the first embodiment. The use of the AST method according to the inventive concept (i.e., based on an analysis of changes in morphology and growth pattern of a microbial cell under antimicrobial agents) makes it possible to obtain accurately and rapidly obtain test results, compared with the AST method in accordance with CLSI guidelines, as will be described with reference to the second embodiment. The use of the AST method according to the inventive concept (i.e., based on an analysis of changes in morphology and growth pattern of a microbial cell under antimicrobial agents) makes it possible to accurately obtain the test results with a faster test time (by about 6-7 times), compared with the standard BMD method in accordance with CLSI guidelines, as will be described with reference to the fourth embodiment. In addition, the use of the AST method according to the inventive concept (i.e., based on an analysis of changes in morphology and growth pattern of a microbial cell under antimicrobial agents) makes it possible to obtain accurate results, even when a microbial cell is grown to exhibit a filament formation and swelling formation, as will be described with reference to the fifth embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

Example embodiments will be more clearly understood from the following brief description taken in conjunction with the accompanying drawings. The accompanying drawings represent non-limiting, example embodiments as described herein.

FIGS. 1(A) and 1(B) illustrate MAC 2.0™ (Quanta Matrix) that was used in a first embodiment. FIG. 1(A) is an image showing a structure of the MAC chip, and FIG. 1(B) shows a cell culturing region.

FIGS. 2(A), 2(B) and 2(C) show MIC characteristics of E. coli ATCC 25922 to antimicrobial agents, according to example embodiments of the inventive concept.

FIG. 2(A) Normal growth (Amikacin): at a concentration lower than MIC, a bacterial cell was divided into two cells. However, at a concentration higher than MIC of Amikacin, there was substantially no division of a bacterial cell.

FIG. 2(B) Filament formation (Piperacillin): at a concentration lower than MIC, a bacterial cell was divided into two cells. At a concentration higher than MIC, there was a formation of a filament (or longitudinal cell expansion) but there was no division of a bacterial cell. In some cases, not only the filament formation but also the cell division was found, and these cases were determined to be resistant.

FIG. 2(C) Cell expansion (Imipenem): at a concentration lower than MIC, a bacterial cell was divided into two cells. At a concentration higher than MIC, a cell was expanded, but there was no division of a bacterial cell. In some cases, not only the swelling formation and the cell division were found, and these cases were determined to be resistant.

FIGS. 3(A), (B) and (C) show MIC characteristics of P. aeruginosa ATCC 27853 to antimicrobial agents, according to example embodiments of the inventive concept.

FIG. 3(A) Normal growth (Gentamicin): at a concentration lower than MIC, a bacterial cell was divided into two cells. However, at a concentration higher than MIC, there was substantially no division of a bacterial cell.

FIG. 3(B) Filament formation (Ceftazidime): at a concentration lower than MIC, a bacterial cell was divided into two cells. At a concentration higher than MIC, a filament was formed, but there was no division of a bacterial cell. In some cases, not only the filament formation but also the cell division was found, and these cases were determined to be resistant.

FIG. 3(C) Cell expansion (Imipenem): at a concentration lower than MIC, a bacterial cell was divided into two cells. However, at a concentration higher than MIC, there was an expansion of a bacterial cell. In some cases, not only the cell expansion and the cell division were found, and these cases were determined to be resistant.

FIGS. 4(A), 4(B) and 4(C) show MIC characteristics of S. aureus ATCC 29213 to antimicrobial agents, according to example embodiments of the inventive concept.

FIG. 4(A) Normal growth (Gentamicin): at a concentration lower than MIC, a bacterial cell was divided into two cells, and thus, the total number of bacterial cells was increased. However, at a concentration higher than MIC, each of bacterial cells was scarcely divided into two cells.

FIG. 4(B) Relatively rapid growth (RRG) (Ciprofloxacin): in the case of Ciprofloxacin, a bacterial cell was rapidly grown, and even at a concentration of MIC or higher, a bacterial cell was rapidly divided into two cells for two hours. However, after four hours, there was no more division of a bacterial cell. It was found that there was a meaningful difference in growth speed between concentrations higher and lower than MIC. The MIC value was determined based on relative growth of a cell.

FIG. 4(C) Relatively slow growth (RSG) (Clindamycin): in the case of clindamycin, although, even at a concentration lower than MIC, a bacterial cell was very slowly divided, there was a meaningful difference in growth speed between concentrations higher and lower than MIC. The MIC value was determined by a relative cell growth.

FIGS. 5(A), (B) and (C) show MIC characteristics of E. faecalis ATCC 29212 to antimicrobial agents, according to example embodiments of the inventive concept.

FIG. 5(A) Normal growth (Vancomycin): at a concentration lower than MIC, each of bacterial cells was divided into two cells, and thus, the total number of the bacterial cells was increased. However, at a concentration higher than MIC, each single bacteria cell was scarcely divided into two cells.

FIG. 5(B) Relatively rapid growth (RRG) (levofloxacin): in the case of levofloacin, even at a concentration of MIC or higher, each of bacterial cells was divided into two cells within two hours, but after four hours, there was no more division of a bacterial cell. It was found that there was a meaningful difference in growth speed between concentrations higher and lower than MIC.

FIG. 5(C) Relatively slow growth (RSG) (linezolid): in the case of linezolid, although, even at a concentration lower than MIC, a bacterial cell was very slowly divided, there was a meaningful difference in growth speed between concentrations higher and lower than MIC. The MIC value was determined based on relative growth of a cell under different concentrations of antimicrobial agents.

FIG. 6 shows results of an antimicrobial susceptibility analysis, according to a first embodiment of the inventive concept. (* Images of gram-positive bacterias were taken at times of 0, 2, and 4 hours. The bar represents 20 μm.)

FIGS. 7(A) and (B) are schematic diagram exemplarily illustrating an image detection method and an antimicrobial susceptibility test method, which are performed using an automated cell image analysis system, according to example embodiments of the inventive concept.

FIGS. 8(A), (B) and (C) shows comparison of MIC characteristics between the AST method according to a fourth embodiment of the inventive concept and the standard method. FIG. 8(A) represents an object strain used for the fourth embodiment of the inventive concept. FIG. 8(B) is a table showing discrepancy rates (DR) and categorical agreement (CA) rates. FIG. 8(C) represents CA rates of clinical strains to β-lactam and non-β-lactam based antimicrobial agents (***p<0.001).

*S: susceptibility; I: intermediate susceptibility; R: resistance; mE: minor error (it is determined as R or S for the standard BMD method and as I for example embodiments of the inventive concept; Or, it is determined as I for the standard BMD method and as R or S for example embodiments of the inventive concept); ME: major error (it is determined as S for the standard BMD method and as R for example embodiments of the inventive concept); VME: very major error (it is determined as R for the standard BMD method and as S for example embodiments of the inventive concept); TTR: the time taken to obtain the measurement.

FIGS. 9(A), (B) and (C) show a reduction of error rate achieved by a method according to a fifth embodiment of the inventive concept, when there are filament and swelling formations (*P<0.05 and ***P<0.001).

FIG. 10 is a diagram to determine whether the microbial cell has antimicrobial susceptibility.

FIG. 11 is a diagram to determine whether the microbial cell has antimicrobial susceptibility.

It should be noted that these figures are intended to illustrate the general characteristics of methods, structure and/or materials utilized in certain example embodiments and to supplement the written description provided below. These drawings are not, however, to scale and may not precisely reflect the precise structural or performance characteristics of any given embodiment, and should not be interpreted as defining or limiting the range of values or properties encompassed by example embodiments. For example, the relative thicknesses and positioning of molecules, layers, regions and/or structural elements may be reduced or exaggerated for clarity. The use of similar or identical reference numbers in the various drawings is intended to indicate the presence of a similar or identical element or feature.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the disclosure(s), specific examples of appropriate materials and methods are described herein. The inventive concept can be variously used depending on the context thereof, and thus, it should not be construed to be limited to a specific one of methods, protocols, and reagents.

As used in the present specification, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. As used in the present specification, the term “or” may have the same meaning as ‘and/or’, unless the context clearly dictates otherwise. It is to be understood that the terms “including”, “comprising”, “consisting” and grammatical variants thereof do not preclude the addition of one or more components, features, steps, integers or groups thereof and that the terms are not to be construed as specifying components, features, steps or integers.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. All documents cited are, in relevant part, incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.

The title of the present specification is provided to express one or more aspects of the inventive concept, but it is not intended to suggest any limitation to the scope of the inventive concept.

Example embodiments of the inventive concept provide antimicrobial susceptibility (AST) methods, which are performed based on an analysis of changes in morphology and growth pattern of a microbial cell under different concentrations of various antimicrobial agents, and an automated cell image analysis system therefor.

According to example embodiments of the inventive concept, an antimicrobial susceptibility (AST) method may be performed based on an analysis of changes in morphology and growth pattern of a microbial cell under antimicrobial agents. For example, the AST method may include the steps of:

-   (a) reacting a microbe with an antimicrobial agent; -   (b) imaging a change in morphology and growth pattern of a microbial     cell over time; -   (c) classifying the change in morphology and growth pattern of a     microbial cell, based on an obtained image; and -   (d) determining resistance and susceptibility of the microbe to the     antimicrobial agent, based on the classification of the change in     morphology and growth pattern of a microbial cell.

In the AST method according to the inventive concept, in step (c), the change in morphology and growth pattern of the microbial cell may be classified into dividing, no-change, filament formation, and swelling formation.

In the AST method according to the inventive concept, in step (d), when the change in morphology and growth pattern of a microbial cell is classified as the dividing, the microbe may be determined to be resistant to the antimicrobial agent, and when the change in morphology and growth pattern of a microbial cell is classified as one of the no-change, the filament formation, and the swelling formation, the microbe may be determined to be susceptible to the antimicrobial agent.

In the present specification, the term “microbe” may refer to all of gram-negative (Gram −) and gram-positive (Gram +) bacteria, Eumycetes, Archimycetes, and so forth, but example embodiments of the inventive concept are not limited thereto. In detail, the microbe may be at least one selected from the group consisting of Enterococcus, Streptococcus, Pseudomonas, Salmonella, Escherichia coli, Staphylococcus, Lactococcus, Lactobacillus, Enterobacteriacae, Klebsiella, Providencia, Proteus, Morganella, Acinetobacter, Burkholderia, Stenotrophomonas, Alcaligenes, and Mycobacterium, but example embodiments of the inventive concept are not limited thereto. In more detail, the microbe may include Enterococcus faecium, Staphylococcus aureus, Klebsiella species, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, but example embodiments of the inventive concept are not limited thereto.

In the present specification, the term “antimicrobial agents” may refer to at least one selected from the group consisting of Amikacin, Amoxicillin, Ampicillin, Aztreonam, Benzylpenicillin, Clavulanic Acid, Cefazolin, Cefepime, Cefotaxime, Cefotetan, Cefoxitin, Cefpodoxime, Ceftazidime, Ceftriaxone, Cefuroxime, Ciprofloxacin, Dalfopristin, Doripenem, Daptomycin, Ertapenem, Erythromycin, Gentamicin, Imipenem, Levofloxacin, Linezolid, Meropenem, Minocycline, Moxifloxacin, Nitrofurantoin, Norfloxacin, Piperacillin, Quinupristin, Rifampicin, Streptomycin, Sulbactam, Sulfamethoxazole, Telithromycin, Tetracycline, Ticarcillin, Tigecycline, Tobramycin, Trimethoprim, and Vancomycin, but example embodiments of the inventive concept are not limited thereto.

In the present specification, the expression “changes in morphology and growth pattern of a microbial cell” may refer to a reaction of a microbial cell to an antimicrobial agent, and such changes of a microbial cell may be classified into dividing, no-change, filament formation, and swelling formation. Here, the expression “changes in morphology and growth pattern of a microbial cell” may be interchangeably used with an expression “morphological change of a microbial cell”.

The term “dividing” may mean that a microbial cell is divided into two cells under non-antibiotic and antibiotic resistant conditions, and consequently, that not only the number of microbial cells but also an OD value of BMD experiment are increased.

The term “no-change” may mean that a microbial cell is susceptible to an antimicrobial agent and is not growing.

The term “filament formation” may mean that a microbial cell is not dividing but is growing in its length direction. The filament formation may include a microbial reaction to β-lactam antimicrobial agents. In detail, the filament formation may include a microbial reaction, except for a reaction of gram-negative bacteria to some (e.g., penems) of β-lactam antimicrobial agents. In more detail, the filament formation may include a microbial reaction, except for a reaction of Pseudomonas aeruginosa or Escherichia coli to some (e.g., penems) of β-lactam antimicrobial agents.

The term “swelling formation” may mean that a microbial cell is not dividing but is swelling and growing. The swelling formation may include a microbial reaction to some (e.g., penems) of β-lactam antimicrobial agents. For example, the swelling formation may include a reaction of gram-negative bacteria to imipenem or meropenem. In detail, the swelling formation may include reactions of Pseudomonas aeruginosa and Escherichia coli to imipenem or meropenem.

The change in morphology and growth pattern of a microbial cell may be observed by imaging a single microbial cell or a group of microbial cells over time.

Accordingly, the AST method according to the inventive concept is be performed based on an analysis of changes in morphology and growth pattern of a microbial cell under antimicrobial agents, and this makes it possible to prevent a microbial cell from dividing. Accordingly, even when an OD value of BMD experiment is constant, it is possible to precisely and rapidly determine the presence or absence of an antimicrobial susceptibility.

According to other example embodiments of the inventive concept, an AST method may be performed based on an analysis of changes in morphology and growth pattern of a microbial cell under antimicrobial agents. For example. The AST method may include the steps of:

-   (a) reacting a microbe with an antimicrobial agent; -   (b) imaging a change in morphology and growth pattern of a microbial     cell over time; -   (c) inspecting an image obtained in step (b) to observe the change     in morphology and growth pattern of a microbial cell; and -   (d) determining an antimicrobial susceptibility of the microbe in     such a way that, when the microbial cell is observed to be in a     state of dividing, the microbial cell is determined to be resistant     to the antimicrobial agent, and when the microbial cell is observed     to be in a state of no-change, filament formation, or swelling     formation, the microbial cell is determined to be susceptible to the     antimicrobial agent.

According to still other example embodiments of the inventive concept, an automated cell image analysis system, which is configured to perform an antimicrobial susceptibility (AST) method, based on an analysis of changes in morphology and growth pattern of a microbial cell under antimicrobial agents, may be provided. For example, the cell image analysis system may include:

-   (a) a culture chip for culture of a microbe and an antimicrobial     agent and for an imaging of a change in morphology and growth     pattern of a microbial cell; -   (b) an optical image analysis device filming a culturing region of     the microbial cell and detecting an image of the microbial cell; and -   (c) a reader analyzing the detected image of the microbial cell to     obtain information on a total area occupied by microbial cells, the     number of microbial cells, and a total length of microbial cells and     determining antimicrobial susceptibility of the microbial cell.

In the automated cell image analysis system, which is configured to perform an antimicrobial susceptibility (AST), based on an analysis of changes in morphology and growth pattern of a microbial cell under antimicrobial agents, according to the inventive concept, the antimicrobial susceptibility may be determined based on a change type (i.e., dividing, no-change, filament formation, and swelling formation) classified according to a total area occupied by microbial cells, the number of microbial cells, and a total length of microbial cells.

With regard to the term “culture chip” used in the present specification, although there is no specific limitation on the type of culture chip, MAC 2.0™ (Quanta Matrix) was used as the culture chip, in the following embodiments.

The term “optical image analysis device” used in the present specification may refer to a device for filming a culturing region of a microbial cell and obtaining an image of the microbial cell and may include a tungsten lamp, an LED device, or a laser light source, which serve as a visible light source for obtaining an optical image. The optical image analysis device may include a charge coupled device (CCD) or complementary metal oxide semiconductor (CMOS) camera for obtaining a cell image transmitted through an objective lens.

The term “reader” used in the present specification may refer to a device for analyzing the obtained image of the microbial cell and determining whether the microbial cell has antimicrobial susceptibility and such operations of the reader may be performed based on a total area occupied by microbial cells, the number of microbial cells, and total length of microbial cells.

In detail, the reader may perform operations of dividing a value (A3) of a total area occupied by microbial cells in a final image (e.g., obtained after 4 hours) by a value (A1) of the total area in an initial image (i.e., at 0 hour), comparing it with a first threshold value (T1), and then

-   a) determining that the microbial cell is resistant, when A3/A1 is     greater than T1, and -   b) performing comparison with a second threshold value (T2) and a     third threshold value (T3), when A3/A1 is smaller than T1, to     determine that, i) if A3/A1 and A3/A2 are greater than each of T2     and T3, the microbial cell is resistant, and ii) otherwise, the     microbial cell is susceptible (here, A2 is a total area occupied by     microbial cells in an image obtained after 2 hours). These     operations may be performed as illustrated in FIG. 10.

Furthermore, when the total area occupied by the microbial cells is increased above the threshold values T1, T2 and T3, the reader may perform as follows. The total length (L3) of the microbial cells in the cell images obtained after 3 hours of cell culture is divided by the number of microbial cells (F3). If L3/F3 is greater than the fourth threshold value (T4), the change is classified as a filament formation or a swelling formation, and the microbial cell is determined to be “susceptible”. If L3/F3 is not greater than T4, the microbial cell is determined to be “resistant”. Here, the threshold values T1, T2, T3 and T4 are individually determined using different growth rates under various antibiotic conditions. More specifically, the reader may perform as follows. The total area (A3) occupied by the microbial cells obtained from a final image after 3 hours of cell culture is divided by the total area (A1) of the microbial cells obtained from an initial image at zero hour, and A3/A1 is compared with the first threshold value (T1). Then the following two steps are performed: (a) in the case that A3/A1 is greater than T1, L3 is divided by F3. If L3/F3 is greater than T4, the change is classified as a filament formation or a swelling formation, and the microbial cell is determined to be susceptible. If L3/F3 is not greater than T4, the microbial cell is determined to be resistant; and (b) in the case that A3/A1 is not greater than T1, the second threshold value (T2) and the third threshold value (T3) are compared with each other and the following steps are conducted: (b1) if A3/A1 is greater than T2 and A3/A2 is greater than T3, wherein A2 is the total occupied areas of microbial cells in the image after 1.5 hours, L3 is divided by F3. If L3/F3 is greater than T4, the change is classified as a filament formation or a swelling formation and the microbial cell is determined to be susceptible. If L3/F3 is not greater than T4, the microbial cell is determined to be resistant; and (b2) if A3/A1 is not larger than T2 or A3/A2 is not larger than T3, the microbial cell is determined to be susceptible. This can be expressed as shown in FIG. 11.

Thus, by using the automated cell image analysis system, which is configured to perform an antimicrobial susceptibility (AST) method using an analysis of changes in morphology and growth pattern of a microbial cell under antimicrobial agents, according to the inventive concept, it is possible to precisely and rapidly examine whether there is an antimicrobial susceptibility, using the total length (L) of microbial cells, even for the cases of filament formation or swelling formation, even when a cell is not dividing and there is no change in the number (F) of microbial cells.

Hereinafter, various embodiments will be exemplarily described to provide a better understanding of the inventive concept. Therefore, the scope of the present inventive concept should not be construed as being limited to the embodiments set forth herein.

Example Embodiments

[FIRST EMBODIMENT] AST Based on an Analysis of Changes in Morphology and Growth Pattern of a Microbial Cell Under Antimicrobial Agents

(1) Objective strain

Four reference strains (E. coli ATCC 25922, S. aureus ATCC 29213, P. aeruginosa ATCC 27853, and E. faecalis ATCC 29212) were used as objective strains, in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines.

(2) Objective antimicrobial agents

To compare antimicrobial susceptibility of E. coli ATCC 25922 and S. aureus ATCC 29213 with that of the standard method, Amikacin, Amoxicillin/Clavulanic Acid, Ampicillin, Aztreonam, Cefazolin, Cefepime, Cefotaxime, Cefoxitin, Ceftazidime, Ciprofloxacin, Gentamicin, Imipenem, Norfloxacin, Meropenem, Piperacillin, Piperacillin/Tazobactam, Tetracycline, Trimethoprim/Sulfamethoxazole, Ticarcilin, Trcarcillin/Clavulanic acid and Tobramycin were used as objective antimicrobial agents, and for P. aeruginosa ATCC 27853 and E. faecalis ATCC 29212, Ampicillin, Amoxicillin/Clavulanic Acid, Ciprofloxacin, Clindamycin, Erythromycin, Gentamicin, Imipenem, Levofloxacin, Linezolid, Oxacillin, Norfloxacin, Penicillin, Rifampin, Streptomycin, Teicoplanin, Tetracycline, Trimethoprim/sulfamethoxazole and Vancomycin were included in the objective antimicrobial agents.

(3) AST based on an analysis of changes in morphology and growth pattern of a microbial cell

MAC 2.0™ (QuantaMatrix) of FIG. 1 was used to immobilize four objective strains, which were prepared to have the standard turbidity, on an agarose well and to inject antimicrobial agents (with a volume of 100 μl and appropriate concentration) into an antimicrobial agent well. The bacterial cell in MAC 2.0™ (QuantaMatrix) was monitored through a S Plan Fluor ELWD 60X (NA 1.49) lens of an inverted optical microscope (Eclipse Ti—Nikon, IX71—Olympus) provided in a heating system, and then, an electron multiplying CCD camera (QuantEM:512SC Photometrics for Eclipse Ti) was used to obtain images of a region where is adjacent to an interface between the agarose well of MAC 2.0™ and the antimicrobial agents well, over time. FIGS. 2 through 5 show the results.

Changes in morphology and growth pattern of the four reference strains (i.e., E. coli ATCC 25922, S. aureus ATCC 29213, P. aeruginosa ATCC 27853, and E. faecalis ATCC 29212) according to the kind of antimicrobial agent can be summarized as follow:

TABLE 1 E. coli ATCC 25922 Criteria Category Antimicrobial Class Amikacin Normal Non-β-lactams aminoglycosides Amoxicillin/ Swelling β-lactams Penicillins/β-lactamase Clavulanic Acid inhibitor Ampicillin Filament β-lactams penicillins Aztreonam Filament β-lactams monobactams Cefazolin Filament β-lactams cephems Cefepime Filament β-lactams cephems Cefotaxime Filament β-lactams cephems Cefoxitin Filament β-lactams cephems Ceftazidime Filament β-lactams cephems Ciprofloxacin Normal Non-β-lactams fluoroquinolone Gentamicin Normal Non-β-lactams aminoglycosides Imipenem Swelling β-lactams penems Norfloxacin Normal Non-β-lactams quinolones Piperacillin Filament β-lactams penicillins Piperacillin/ Filament β-lactams Penicillins/β-lactamase Tazobactam inhibitor Tetracycline Normal Non-β-lactams tetracyclines Trimethoprim/ Normal Non-β-lactams folate pathway Sulfamethoxazole inhibitors

TABLE 2 P. aeruginosa ATCC 27853 Criteria Category Antimicrobial Class Amikacin Normal Non-β-lactams aminoglycosides Aztreonam Filament β-lactams monobactams Cefepime Filament β-lactams cephems Cefotaxime Filament β-lactams cephems Ceftazidime Filament β-lactams cephems Ciprofloxacin Normal Non-β-lactams fluoroquinolone Gentamicin Normal Non-β-lactams aminoglycosides Imipenem Swelling β-lactams penems Meropenem Swelling β-lactams penems Piperacillin Filament β-lactams penicillins Piperacillin/Tazobactam Filament β-lactams Penicillins/ β-lactamase inhibitor Ticarcilin Filament β-lactams penicillins Ticarcillin/Clavulanic acid Filament β-lactams Penicillins/ β-lactamase inhibitor tobramycin Normal Non-β-lactams aminoglycosides

TABLE 3 S. aureus ATCC 29213 Criteria Category Antimicrobial Class Ampicillin Normal β-lactams penicillins Amoxicillin/ Normal β-lactams Penicillins/ Clavulanic Acid β-lactamase inhibitor Ciprofloxacin RRG Non-β-lactams fluoroquinolone Clindamycin RSG Non-β-lactams lincosamides erythromycin RSG Non-β-lactams macrolides Gentamicin Normal Non-β-lactams aminoglycosides Imipenem Normal β-lactams penems Levofloxacin RRG Non-β-lactams quinolones Linezolid RSG Non-β-lactams oxazolidinones Oxacillin RRG β-lactams penicillins Penicillin Normal β-lactams penicillins Rifampin RSG Non-β-lactams ansamycins Tetracycline Normal Non-β-lactams tetracyclines Trimethoprim/ RRG Non-β-lactams folate pathway sulfamethoxazole inhibitors Vancomycin normal Non-β-lactams glycopeptides

TABLE 4 E. faecalis ATCC 29212 Criteria Category Antimicrobial Class Ampicillin Normal β-lactams penicillins Ciprofloxacin RRG Non-β-lactams fluoroquinolone erythromycin RSG Non-β-lactams macrolides Gentamicin High Level Normal Non-β-lactams aminoglycosides Levofloxacin RRG Non-β-lactams quinolones Linezolid RSG Non-β-lactams oxazolidinones Norfloxacin RRG Non-β-lactams quinolones Penicillin Normal β-lactams penicillins Rifampin RSG Non-β-lactams ansamycins Streptomycin High Level Normal Non-β-lactams aminoglycosides Teicoplanin Normal Non-β-lactams glycopeptides Tetracycline RSG Non-β-lactams tetracyclines Vancomycin Normal Non-β-lactams glycopeptides

(4) Results

A summary of AST results obtained from changes in morphology and growth pattern of the objective strains is illustrated in FIG. 6. Referring to FIG. 6, when the antimicrobial susceptibility is determined by the standard AST method per CLSI guidelines, a sample illustrated in a portion (A) of FIG. 6 was determined to be resistance to the antimicrobial agents, because an OD value obtained by the BMD method was increased, whereas samples illustrated in portions B, C, and D of FIG. 6 were determined to be susceptible to the antimicrobial agents, because the OD values obtained by the BMD method were decreased. In the cases of portions C and D of FIG. 6, there was no dividing and cells were growing, but the samples were determined to be susceptible; that is, it was found that the standard AST method gives an inaccurate AST result.

By contrast, when the antimicrobial susceptibility is determined by the AST method according to the inventive concept (i.e., based on an analysis of changes in morphology and growth pattern of a microbial cell), the sample illustrated in the portion (A) of FIG. 6 was determined to be resistance to the antimicrobial agents, because the morphological pattern thereof was classified as a case of dividing, and the sample illustrated in the portion (B) of FIG. 6 was determined to be susceptible to the antimicrobial agents, because the morphological pattern thereof was classified as a case of no-change. The sample illustrated in the portion (C) of FIG. 6 was determined to be susceptible to the antimicrobial agents, because the morphological pattern thereof was classified as a case of filament formation, and the sample illustrated in the portion (D) of FIG. 6 was determined to be susceptible to the antimicrobial agents, because the morphological pattern thereof was classified as a case of swelling formation. That is, it was found that the AST method according to the inventive concept gives a more accurate AST result, compared with that of the conventional AST method.

[SECOND EMBODIMENT] Evaluation on Accuracy and Rapidity of the AST Method According to the Inventive Concept

In the AST method according to the inventive concept, minimal inhibitory concentration (MIC) was measured using the changes in morphology and growth pattern of the four reference strains (i.e., E. coli ATCC 25922, S. aureus ATCC 29213, P. aeruginosa ATCC 27853, and E. faecalis ATCC 29212) and was compared with MIC quality control (QC) per CLSI guidelines. The following table 5 shows the results.

TABLE 5 Comparison of MIC values according to proposed method and QC of CLSI Gram Negative Strains E. coli P. aeruginosa ATCC 25922 ATCC 27853 SCMA CLSI SCMA CLSI Antimicrobial Results QC range Results QC range Amikacin 0.5~1   0.5~4   1~2 1~4 Amoxicillin/Clavulanic Acid 4/2 2/1~8/4 — — Ampicillin 2~4 2~8 — — Aztreonam 0.12 0.06~0.25 2~4 2~8 Cefazolin 2 1~4 — — Cefepime 0.03~0.06 0.015~0.12  0.5~2   0.5~4   Ceforaxime 0.06 0.03~0.12 16  8~32 Cefoxitin 2 2~8 — — Ceftazidime 0.5 0.06~0.5  2~4 1~4 Ciprofloxacin 0.004~0.008 0.004~0.015 0.25~0.5  0.25~1   Gentamicin 0.25 0.25~1   0.5~1   0.5~2   Imipenem 0.12 0.06~0.25 1 1~4 Norfloxacin 0.03~0.06 0.03~0.12 — — meropenem — — 1 0.25~1   Piperacillin 2 1~4 4~8 1~8 Piperacillin/Tazobactam 1/4~2/4 1/4~4/4 2/4~8/4 1/4~8/4 Tetracycline 1 0.5~2   — — Trimethoprim/Sulfamethoxazole ≤0.5/9.5    ≤0.5/9.5 — — Ticarcilin — — 16  8~32 Ticarcillin/Clavulanic acid — — 16/2  8/2~32/2 Tobramycin — — 0.25~0.58 0.25~1   Time to Result 3 hr 16~20 hr 3 hr 16~20 hr Gram Positive Strains S. aureus E. faecalis ATCC 29213 ATCC 29212 SCMA CLSI SCMA CLSI Antimicrobial Results QC range Results QC range Ampicillin 1 0.5~2   0.5~1   0.5~2   Amoxicillin/Clavulanic Acid 0.25/0.12~0.5/0.25  0.12/0.06~0.5/0.25  — — Ciprofloxacin 0.5 0.12~0.5  0.5~2   0.25~2   Clindamycin 0.12 0.06~0.25 — — Erythromycin 0.25 0.25~1   1 (6 hr) 1~4 Gentamicin 0.25~0.5  0.12~1   ≤500 ≤500 Imipenem 0.03 0.015~0.06  — — Levofloxacin 0.12~0.25 0.06~0.5  1 0.25~2   Linezolid 1~4 1~4 1~2 1~4 Oxacillin 0.5 0.12~0.5  — — Norfloxacin — — 2~4 2~8 Penicillin 0.5 0.25~2   1~2 1~4 Rifampin 0.004~0.08 (6 hr) 0.004~0.015 0.5 0.5~4   Streptomycin High Level — — ≤500 ≤500 Teicoplanin — — 0.5 0.25~1   Tetracycline 0.12~0.5  0.12~1   8 (6 hr)  8~32 Trimethoprim/sulfamethoxazole ≤0.5/9.5 ≤0.5/9.5 — — Vancomycin 1 0.5~2 2~4 1~4 Time to Result 4 hr 16~20 hr 4 hr 16~20 hr

Table 5 shows that MIC values obtained by the method according to the inventive concept (i.e., based on an analysis of changes in morphology and growth pattern of a microbial cell) were included within the MIC QC range in accordance with CLSI guidelines. In addition, it generally takes 16-20 hours to obtain the MIC results in accordance with CLSI guidelines, whereas it takes about 3-4 hours to obtain the MIC results using the method according to the inventive concept. That is, it was found that by using the AST method according to the inventive concept, it was possible to rapidly provide accurate data, compared with the conventional AST method in accordance with CLSI guidelines.

[THIRD EMBODIMENT] AST Method Using an Automated Cell Image Analysis System

An automated image analysis program was coded with MATLAB R2013a (MathWorks). A raw image of RGB format was processed to remove noises therefrom and obtain an image of binary format showing features and background of bacteria. Pixel information associated with the features of bacteria in the image was calculated to obtain information on the region, number and length of bacterial cells. The results are shown in FIG. 7.

[FOURTH EMBODIMENT] Comparison of MIC Values According to the Proposed AST Method and the Standard Method

(1) Objective strain

189 strains, which had been isolated from various clinical specimens of patients at Seoul National University Hospital, Korea (SNUH, 149 strains) and Incheon St. Mary's Hospital (ISMH, 40 strains) (42 E. coli, 34 P. aeruginosa, 30 K. pneumoniae, 45 S. aureus, and 38 Enterococcus spp.) were used as the objective strain.

(2) Method of Experiment

The isolated clinical strains were grown in a sheep blood agar medium or a Mueller Hinton Agar (MHA) medium. Before the experiment, each of the strains was subcultured in a cation-adjusted MHB (CAMHB) medium for 20-24 hours. Thereafter, the AST method according to the inventive concept was performed at the same time as the standard BMD method in accordance with CLSI guidelines that was performed as a comparative experiment.

(3) Comparison of MICs and results

AST results according to the inventive concept were compared with results obtained by the standard BMD method. The results obtained by the comparison were categorized into categorical agreement (CA), minor error (mE), very major error (VME), and major error (ME), in accordance with U.S. Food and Drug Administration Guidance. For example, in the case that the AST results according to the inventive concept coincided with decision by the standard BMD method, the comparison result was defined as categorical agreement (CA). In the case that one of the results obtained by two methods was intermediate and the other was resistant or susceptible, the comparison result was defined as minor error (mE). In the case that the result of the standard BMD method was resistant and the result of the AST method was susceptible, the comparison result was defined as very major error (VME). In the case that the result of the standard BMD method was susceptible and the result of the AST method was resistant, the comparison result was defined as major error (ME). The results are shown in FIG. 8.

Referring to FIG. 8, when the AST according to the inventive concept was used, a rate of CA was 91.5%, and rates of mE, ME, and VME were 6.3%, 2.9%, and 1.4% respectively, and this satisfied the criteria suggested by FDA, which sets the standard for the minimal performance requirements (mE 10%, ME 3.0%, VME 1.5%, CA 90%). Also, low CA was obtained in reactions of gram-negative strains to β-lactam antimicrobial agents, and there were no significant differences between β-lactam and non β-lactam antimicrobial agents.

Accordingly, these results shows that the AST method according to the inventive concept (i.e., based on analysis of changes in morphology and growth pattern of a microbial cell) made it possible to accurately obtain the test results with a faster test time (by about 6-7 times), compared with the standard BMD method in accordance with CLSI guidelines.

[FIFTH EMBODIMENT] Evaluation of Error Rate Associated with the Filament and Swelling Formations, in the AST Method According to the Inventive Concept

The graph of FIG. 9 shows minor error rate, major error rate, and very major error rate, in the above experiment (i.e., according to the fourth embodiment) for determining susceptibilities of gram-negative strain E. coli, P. aeruginosa, and K. Pneumonia to β-lactam and non-β-lactam antimicrobial agents.

Referring to FIG. 9, in the case of the reaction with β-lactam antimicrobial agents, the major error rate was abruptly decreased (E. coli: from 12.8% to 3.7% and P. aeruginosa: from 48.1% to 9.9%). This means that, for the reaction between the gram-negative strain and the β-lactam antimicrobial agents, the result obtained by the standard BMD method was determined to be resistant. However, when the AST method according to the inventive concept was used, the result was determined to be susceptible for the filament formation, and this made it possible to reduce the major error rate. In addition, it was found that, for the reaction between E. coli and non-β-lactam antimicrobial agents, the use of the AST method according to the inventive concept made it possible to reduce the major error rate.

While the present general inventive concept has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present general inventive concept as defined by the following claims. The preferred embodiments should be considered in descriptive sense only and not for purposes of limitation. Therefore, the scope of the invention is defined not by the detailed description of the invention but by the appended claims, and all differences within the scope will be construed as being included in the present invention. 

What is claimed is:
 1. An automated cell image analysis system configured to perform an antimicrobial susceptibility (AST) method based on an analysis of changes in morphology and growth pattern of a microbial cell under antimicrobial agents, wherein the cell image analysis system comprises: (a) a culture chip for culture of a microbe antimicrobial cells and an antimicrobial agent and for an imaging of a change in morphology and growth pattern of a microbial cell; (b) an optical image analysis device filming a culturing region of the microbial cell and detecting an image of the microbial cell; and (c) a reader analyzing the detected image of the microbial cell to obtain information on a total area occupied by microbial cells, the number of microbial cells, and a total length of microbial cells and determining antimicrobial susceptibility of the microbial cell.
 2. The cell image analysis system of claim 1, wherein the change in morphology and growth pattern of the microbial cells is classified into dividing, no-change, filament formation, and swelling formation, according to the total area occupied by microbial cells, the number of microbial cells, and the total length of microbial cells, and the antimicrobial susceptibility is determined based on the classification of the change in morphology and growth pattern of the microbial cells.
 3. The cell image analysis system of claim 1, wherein the optical image analysis device comprises a tungsten lamp, a LED device, or a laser light source.
 4. The cell image analysis system of claim 1, wherein the optical image analysis device comprises a charge coupled device (CCD) or complementary metal oxide semiconductor (CMOS) camera.
 5. The cell image analysis system of claim 1, wherein the reader performs steps of: dividing a value A3, which represents the total area occupied by microbial cells in a final image obtained after 4 hours, by a value A1, which represents the total area in an initial image at 0 hour; performing comparison with a first threshold value T1; determining that the microbial cell is resistant, when A3/A1 is greater than T1, and performing comparison with a second threshold value T2 and a third threshold value T3, when A3/A1 is smaller than T1, to determine that, i) if A3/A1 and A3/A2 are greater than each of T2 and T3, the microbial cell is resistant, and ii) otherwise, the microbial cell is susceptible, where A2 is a total area occupied by microbial cells in an image obtained after 2 hours.
 6. The cell image analysis system of claim 1, wherein the reader performs steps of: dividing a value A3, which is the total area occupied by the microbial cells in a final image obtained after 3 hours, by a value A1, which is the total area of the microbial cells obtained from an initial image at zero hour; comparing A3/A1 with a first threshold value T1 and performing the following steps: (a) in the case that A3/A1 is greater than T1, dividing a value L3, which is the total length of the microbial cells in the final image, by a value F3, which is the number of the microbial cells; comparing L3/F3 with a fourth threshold value T4; and determining that: if L3/F3 is greater than T4, the change in morphology and growth pattern of the microbial cells are classified as a filament formation or a swelling formation and the microbial cells are susceptible to the antimicrobial agent; and if L3/F3 is not greater than T4, the microbial cells are resistant to the antimicrobial agent; and (b) in the case that A3/A1 is not greater than T1, performing the following steps: (b1) dividing L3 by F3 if A3/A1 is greater than a second threshold value T2 and A3/A2 is greater than a third threshold value T3, wherein A2 is total area occupied by the microbial cells in the image after 1.5 hours; and determining that: if L3/F3 is greater than T4, the change in morphology and growth pattern of the microbial cells is classified as a filament formation or a swelling formation and the microbial cell is susceptible to the antimicrobial agent; and if L3/F3 is not greater than T4, the microbial cells are resistant to the antimicrobial agent; and (b2) determining that the microbial cells are susceptible to the antimicrobial agent if A3/A1 is not greater than T2 or A3/A2 is not greater than T3. 